The present invention relates to novel guanosine triphosphate binding protein-coupled receptor proteins, DNA encoding the proteins, and methods of screening for drug-candidate compounds using them.
Many hormones and neurotransmitters regulate physiological functions through specific receptor proteins located on the cell membrane. Many of these receptor-proteins transduce signals into the cell by activating a guanosine triphosphate binding protein (occasionally referred to as xe2x80x9cG proteinxe2x80x9d below) that is coupled to them. These receptor proteins are thereby named as G protein-coupled receptors. Since they have a common structure, composed of seven transmembrane regions, they are also generally called xe2x80x9cseven-transmembrane receptor proteins.xe2x80x9d
G protein-coupled receptors, which are expressed on the surface of cells in vivo and functioning cells of tissues, play an extremely important role as a target of molecules such as hormones, neurotransmitters, and biologically active compounds, which regulate the functions of these cells and tissues. Therefore, G protein-coupled receptor proteins have received great attention as targets in drug-development.
G protein-coupled receptors reported so far include: muscarinic acetylcholine receptors M1, M2, M3, and M4 (Peralta et al., EMBO J., 6:3923-3929 (1987)), muscarinic acetylcholine receptor M5 (Bonner et al., Neuron, 1:403-410 (1988)), adenosine receptor A1 (Libert et al., Science, 244:569-572 (1989)), xcex11A adrenoreceptor (Bruno et al., Biochem. Biophys. Res. Commun., 179:1485-1490 (1991)), xcex21 adrenoreceptor (Frielle et al., Proc. Natl. Acad. Sci. USA, 84:7920-7924 (1987)), angiotensin receptor AT1 (Takayanagi et al., Biochem. Biophys. Res. Commun., 183:910-916 (1992)), endothelin receptor ETA (Adachi et al., Biochem. Biophys. Res. Commun., 180:1265-1272 (1991)), gonadotropin releasing factor receptor (Kaker et al., Biochem. Biophys. Res. Commun., 189:289-295 (1992)), histamine receptor H2 (Ruat et al., Proc. Natl. Acad. Sci. USA, 87:1658-1672 (1992)), neuropeptide Y receptor Y1 (Larhammar et al., J. Biol. Chem., 267:10935-10938 (1992)), interleukin-8 receptor IL8RA (Holmes et al., Science, 2563:1278-1280 (1991)), dopamine receptor D1 (Mahan et al., Proc. Natl. Acad. Sci. USA, 87:2196-2200 (1990)), metabolic glutamate receptor mGluR/1 (Masu et al., Nature, 349:760-765 (1991)), and somatostatin receptor SS1 (Yamada et al., Proc. Natl. Acad. Sci. USA, 89:251-255) (for reference, Watson S. and Arkinstall S., The G protein Linked Receptor FactsBook, Academic Press (1994)). Examples of developed medicines aimed at G protein-coupled receptors are: terazosine hydrochloride (antihypertensive agent, xcex11 adrenoreceptor antagonist), atenolol (antiarrhythmia, xcex21 adrenoreceptor antagonist), dicyclomine hydrochloride (antispasmodic agent, acetylcholine receptor antagonist), ranitidine hydrochloride (drug for peptic ulcers, histamine receptor H2 antagonist), trazodone hydrochloride (antidepressant, serotonin receptor 5-HT1B antagonist), and buprenorphine hydrochloride (analgesic agent, opioid receptor xcexa agonist) (for reference, Stadel et al., Trends Pharm. Sci., 18:430-437 (1997); Medicine Handbook 5th edition, Yakugyo-Jiho).
The hypothalamus, a part of the brain which governs a number of programs that trigger a particular response, contributes to the homeostasis of the internal environment by means of a variety of outputs, as the center of the autonomic nervous system. For instance, it releases hormones such as thyrotropic hormone-releasing hormone, gonadotropic hormone-releasing hormone, and growth hormone-releasing hormone, and thereby regulates the entire endocrine system through the actions of these hormones on the specific receptors expressed in target cells. These outputs in the hypothalamus are thought to be mediated by receptors expressed in the hypothalamus and compounds reacting with them. Therefore, elucidation of the relationship between the compounds regulating the hypothalamus outputs and their specific receptors expressed in the hypothalamus is extremely important in developing novel medicines for the treatment of diseases arising from endocrine disorders.
The present invention provides a novel human-derived G protein-coupled receptor protein and rat-derived one corresponding thereto, both of which are expressed in the brain (in particular, thalamus and hypothalamus, etc.). It also provides a method of screening for ligands and drug-candidate compounds using these receptor proteins.
The inventors first selected a region highly conserved in known G protein-coupled receptor proteins, then designed primers corresponding to the region, and performed reverse transcriptase-polymerase chain reaction (RT-PCR) using mRNA obtained from rat thalamus and hypothalamus. Next, amplified clones were randomly selected, and their partial nucleotide sequences were determined. To remove known clones from the nucleotide sequence determined-clones, colony-hybridization was performed using, as a probe, cDNA clones judged by homology search to be encoding a known G protein-coupled receptor protein. Negative clones that failed to hybridize with any probe were selected. Using probes prepared based on the nucleotide sequence of the negative clones, the inventors screened cDNA libraries from rat thalamus and hypothalamus, and succeeded in isolating a full-length cDNA (rat BG2 cDNA) encoding a rat G protein-coupled receptor.
Moreover, the present inventors screened human hippocampus libraries using specific probes and successfully isolated a human cDNA (human BG2 cDNA) corresponding to the rat cDNA.
To identify a ligand for the G protein-coupled receptor protein encoded by the isolated human BG2 cDNA, the present inventors prepared cells expressing the protein and stimulated the protein to screen compounds which changed the concentration of cAMP in the cells. As a result, histamine was found to have an activity of stimulating the human G protein-coupled receptor protein expressed on the cell surface and lowering the intracellular cAMP concentration. In addition, histamine was found to have an activity of actually binding to the protein.
The present inventors also isolated a cDNA encoding an alternative splicing variant for rat BG2 (rat BG2-2 cDNA), expressed the protein encoded by the cDNA on a cell surface, and detected the response against the histamine stimulation, to find that the protein had an activity of reducing the intracellular cAMP concentration in response to the histamine stimulation in the same manner as in human BG2.
The G protein-coupled receptor protein found by the present inventors is a very useful tool in screening for agonists and antagonists thereof, and the agonists and antagonists isolated by the screening are expected to be used as pharmaceuticals.
The present invention relates to novel human- and rat-derived G protein-coupled receptor proteins, DNAs encoding them, and screening of ligands and drug-candidate compounds using the proteins.
Specifically, the invention relates to:
(1) a guanosine triphosphate-binding protein-coupled receptor protein comprising the amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:20 or 25, and
(b) the amino acid sequence of SEQ ID NO:20 or 25, in which one or more amino acids are replaced, deleted, or added;
(2) the protein of (1), wherein the protein has an activity of binding to histamine;
(3) the protein of (1), wherein the protein has an activity of changing the intracellular cAMP concentration or calcium concentration in response to histamine stimulation;
(4) a partial peptide of the receptor protein of any one of (1) to (3);
(5) a DNA encoding the receptor protein of any one of (1) to (3) or the partial peptide of (4);
(6) the DNA of (5), wherein the DNA comprises a coding region of the nucleotide sequence of SEQ ID NO:21 or 26;
(7) a vector containing the DNA of (5) or (6);
(8) a transformant carrying the DNA of (5) or (6) or the vector of (7);
(9) a method for producing the receptor protein of any one of (1) to (3) or the partial peptide of (4), the method comprising the steps of culturing the transformant of (8) and recovering the protein or peptide from the transformant or its culture supernatant;
(10) a method of screening for a ligand which binds to the receptor protein of any one of (1) to (3), or an analogue thereof, the method comprising the steps of:
(a) exposing a test compound to the receptor protein of any one of (1) to (3) or the partial peptide of (4), and
(b) selecting the compound that binds to the protein or partial peptide;
(11) a method of screening for a compound that inhibits the binding between the receptor protein of any one of (1) to (3) and its ligand or an analogue of the ligand, the method comprising the steps of:
(a) exposing a ligand or its analogue to the receptor protein of any one of (1) to (3) or the partial peptide of (4) in the presence of a test compound, and detecting the binding activity between the protein or partial peptide and the ligand or its analogue, and
(b) comparing the binding activity detected in (a) with that in the absence of the test compound, and selecting the compound that reduces the binding activity;
(12) the method of (11), wherein the ligand is histamine;
(13) a method of screening for a compound which inhibits or promotes the activity of the receptor protein of any one of (1) to (3), the method comprising the steps of:
(a) exposing a ligand for the protein or an analogue thereof to cells expressing the protein in the presence of a test compound,
(b) detecting a change in cells associated with the binding of the protein to the ligand or the analogue thereof, and
(c) selecting a compound which inhibits or promotes the change in the cells detected in (b) in comparison with the change in the cells in the absence of the test compound;
(14) the method of (13), wherein the ligand is histamine;
(15) the method of (13) or (14), wherein the change in cells to be detected is selected from the group consisting of a change in cAMP concentration, a change in calcium concentration, an activation of G protein, an activation of phospholipase C, and a change in pH;
(16) a kit for the method of any one of (10) to (15), the kit comprising the receptor protein of any one of (1) to (3) or the partial peptide of (4);
(17) an antibody which binds to the receptor protein of any one of (1) to (3);
(18) a compound isolated by the method of any one of (11) to (15); and
(19) a pharmaceutical composition comprising the compound of (18) as an active ingredient.
xe2x80x9cG protein-coupled receptor proteinxe2x80x9d herein refers to a receptor protein that transduces intracellular signals by activating G proteins.
xe2x80x9cLigandxe2x80x9d refers to a natural compound capable of binding to a G protein-coupled receptor and inducing signal transduction. xe2x80x9cAnalogue of a ligandxe2x80x9d herein refers to a derivative of the ligand having the same physiological activity as the ligand binding to a G protein-coupled receptor protein, or inhibiting a physiological activity of the ligand, and contains both natural and artificially synthesized compounds. For example, histamine and R(xe2x88x92)-xcex1-methylhistamine correspond to a ligand and an analogue thereof, respectively.
xe2x80x9cAgonistxe2x80x9d refers to a compound having a bioactivity similar to that of the ligands of G protein-coupled receptors, including both natural and artificially synthesized compounds.
xe2x80x9cAntagonistxe2x80x9d refers to a compound capable of inhibiting the bioactivity of a ligand of a G protein-coupled receptor, including both natural and artificially synthesized compounds.
xe2x80x9cProteinxe2x80x9d and xe2x80x9cpeptidexe2x80x9d as used herein include their salts as well.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different DNA molecules, transfected cells, or cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. For example, the substantially pure polypeptide is at least 75%, 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
The present invention provides novel G protein-coupled receptor proteins derived from human and rat. The nucleotide sequence of the cDNA encoding human G protein-coupled receptor (human BG2) isolated herein is shown in SEQ ID NO:21, and the amino acid sequence of the human BG2 protein is shown in SEQ ID NO:20. The nucleotide sequence of the cDNA encoding rat G protein-coupled receptor (rat BG2-2) isolated in the present invention is shown in SEQ ID NO:26, and the amino acid sequence of the rat BG2-2 protein is shown in SEQ ID NO:25.
Human BG2 protein has 32%, 28%, and 27% homology to known G protein-coupled receptors, namely human xcex1-2C-1 adrenoreceptor (Regan et al., Proc. Natl. Acad. Sci. USA, 85:6301-6305 (1988)), mouse xcex2-1 adrenoreceptor (Jasper et al., Biochim. Biophys. Acta., 1178:307-309 (1993)), and human muscarinic acetylcholine receptor M3 protein (Peralta et al., EMBO J., 6:3923-3929 (1987)), respectively. These results suggest that the human BG2 proteins belong to the G protein-coupled receptor family. Furthermore, this suggests that they participate in signal transduction through the activation of G proteins upon ligand binding. In fact, human BG2 protein has an activity of binding to histamine, and of reducing the intracellular cAMP concentration in response to the stimulation by histamine. Human BG2 protein of the present invention was expressed in the brain (for example, hippocampus). In brain, hippocampus plays an important role in memory and learning, the cerebellum regulates the body motions, and the hypothalamus serves as the center of the autonomic nervous system. Thus, the human BG2 proteins are assumed to be involved in the regulation of these functions. Therefore, the proteins and genes, or an agonist or antagonist that can regulate the human BG2 protein function(s), can be used in the treatment of disabilities in memory and learning, or the control of the autonomous nervous system, such as regulation of blood pressure, digestion, body temperature, food-intake, etc. In addition, rat BG2-2 protein is a rat protein corresponding to human BG2 protein and is, like human BG2 protein, a G protein-coupled receptor protein whose ligand is histamine.
The proteins of the present invention may be prepared as natural proteins, and also as recombinant proteins, by using recombinant DNA technology. A natural protein may be prepared, for instance, by extracting tissues, speculated to express the protein of the present invention, and performing immunoaffinity chromatography using antibody as described later on. On the other hand, a recombinant protein can be prepared by culturing transformant cells carrying DNA encoding the protein of the present invention as described later on. One skilled in the art can prepare an altered protein having a function or an activity (transduction of intracellular signals through G protein activation, binding activity to histamine, and activity of varying concentration of intracellular cAMP or calcium responded by histamine stimulation) equivalent to that of the natural protein by introducing modifications such as replacement of any amino acid contained in the natural protein of the present invention (SEQ ID NO:20 or SEQ ID NO:25) according to known methods. Mutations of amino acids in a protein may occur naturally. The G protein-coupled receptor proteins of the present invention include such mutant proteins having an amino acid sequence altered by replacement, deletion or addition, having a function equivalent to that of the natural protein. The methods of altering amino acids, known to one skilled in the art, include, the Kunkel method (Kunkel et al., Methods Enzymol., 154:367-382 (1987)), double primer method (Zoller et al., Methods Enzymol., 154:329-350 (1987)), cassette mutation (Wells et al., Gene, 34:315-323 (1985)), and megaprimer method (Sarkar et al., Biotechniques, 8:404-407 (1990)). The number of mutated amino acids in a functionally equivalent protein is generally not more than 10% of all the amino acids, preferably not more than 10 amino acids, and more preferably not more than 3 amino acids (for instance, one amino acid).
The invention also includes a polypeptide, or fragment thereof, that differs from the corresponding sequence shown as SEQ ID NO:20 or SEQ ID NO:25. The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In one embodiment, the polypeptide includes an amino acid sequence at least about 60% identical to a sequence shown as SEQ ID NO:20 or SEQ ID NO:25, or a fragment thereof. Preferably, the polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:20 or SEQ ID NO:25 and has at least one G-protein coupled receptor protein function or activity described herein. Preferred polypeptide fragments of the invention are at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of the sequence shown as SEQ ID NO:20 or SEQ ID NO:25 and have at least one G-protein coupled receptor protein activity described herein. Or alternatively, the fragment can be merely an immunogenic fragment.
As used herein, xe2x80x9c% identityxe2x80x9d of two amino acid sequences, or of two nucleic acid sequences, is determined using the algorithm of Karlin and Altschul (PNAS USA, 87:2264-2268, 1990), modified as in Karlin and Altschul, PNAS USA, 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol., 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignment for comparison purposes GappedBLAST is utilized as described in Altschul et al (Nucleic Acids Res., 25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention.
The present invention also includes partial peptides of the above-described G protein-coupled receptor proteins. The partial peptides of the present invention include, for instance, those corresponding to the N-terminal region of the G protein-coupled receptor protein, which can be utilized to prepare an antibody. Moreover, partial peptides of the present invention include peptides having the binding activity to histamine and peptides having an activity of changing the intracellular cAMP concentration or calcium concentration in response to the stimulation by histamine when expressed on the cell surface. These peptides can be used for screening drug-candidate compounds as described below. Moreover, a partial peptide which has the binding activity to histamine but does not have an activity of conducting the signal transduction into cells, can be a competitive inhibitor for the proteins of the present invention. Such partial peptides of the invention usually have a length of at least 15 amino acids, and preferably 20 amino acids or more.
Furthermore, the present invention provides DNA encoding the proteins of the invention as described above or partial peptides thereof. The DNA encoding the protein of the invention or partial peptide thereof include cDNA, genomic DNA, and synthetic DNA, but are not so limited as long as they encode the proteins or the peptides. cDNA encoding the proteins of the present invention can be screened by labeling, with 32P or the like, for example, the cDNA as described in SEQ ID NO:21 or NO:26, a part of it, complementary RNA to the DNA, or a synthetic oligonucleotide comprising a part of the cDNA and by hybridizing them to a cDNA library from a tissue expressing the protein of the present invention (for instance, brain tissue). Alternatively, cDNA may be cloned by synthesizing an oligonucleotide corresponding to the nucleotide sequence of the cDNA, and amplifying cDNA from an appropriate tissue (such as brain tissue) by PCR. Genomic DNA can be obtained by screening a genomic library by hybridization using, as a 32P-labeled probe, the cDNA as described in SEQ ID NO:21 or NO:26, or a part of it, complementary RNA to the DNA, or a synthetic oligonucleotide comprising a part of the cDNA. Alternatively, it may be cloned by synthesizing an oligonucleotide corresponding to the nucleotide sequence of the cDNA, and amplifying genomic DNA by PCR. Synthetic DNA can be prepared by chemically synthesizing oligonucleotides comprising a part of the nucleotide sequence of SEQ ID NO:21 or NO:26, annealing them into a double strand, and ligating them using DNA ligase (Khorana et al., J. Biol. Chem., 251:565-570 (1976); Goeddel et al., Proc. Natl. Acad. Sci. USA, 76:106-110 (1979)).
In one aspect, the invention provides an isolated or purified nucleic acid molecule that encodes a polypeptide described herein or a fragment thereof. Preferably, the isolated nucleic acid molecule includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO:21 or SEQ ID NO:26. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ ID NO:21 or SEQ ID NO:26. In the case of an isolated nucleic acid molecule which is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO:21 or SEQ ID NO:26, the comparison is made with the full length of the reference sequence. Where the isolated nucleic acid molecule is shorter that the reference sequence, e.g., shorter than SEQ ID NO:21 or SEQ ID NO:26, the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).
The invention also includes nucleic acid sequences that hybridize to the nucleic acid molecule shown as SEQ ID NO:21 or SEQ ID NO:26, or a fragment thereof. Hybridization is performed in 6xc3x97SSC, 40% formamide at 25xc2x0 C., followed by a wash in 1xc3x97SSC at 55xc2x0 C. (low stringency); in 6xc3x97SSC, 40% formamide at 37xc2x0 C., followed by a wash in 0.2xc3x97SSC at 55xc2x0 C. (medium stringency); or in 6xc3x97SSC, 40% formamide at 37xc2x0 C., followed by a wash in 0.1xc3x97SSC at 62xc2x0 C. (high stringency).
These DNA can be used for producing recombinant proteins. Namely, it is possible to prepare the protein of the invention as a recombinant protein by inserting a DNA encoding the receptor protein (DNA as described in SEQ ID NO:21 or NO:26, for instance) into an appropriate expression vector, culturing a transformant obtained by introducing the vector into an appropriate cell, and purifying the expressed protein. Since the protein of the invention is a receptor protein, it is possible to prepare it in a form expressed on the cell membrane.
Specifically, if the host is Escherichia coli, the plasmid vectors such as pET-3 (Rosenburg et al., Gene, 56:125-135 (1987)) and pGEX-1 (Smith et al., Gene, 67:31-40 (1988)) may be used. E. coli can be transformed by the Hanahan method (Hanahan D., J. Mol. Biol., 166:557-580 (1983)), electroporation (Dower et al., Nucleic Acids Res., 16:6127-6145 (1988)), and such. If the host is fission yeast (Schizosaccharomyces pombe), a plasmid vector such as pESP-1 (Lu et al., Gene, 200:135-144 (1997)) may be used. Yeast can be transformed by spheroplast fusion (Beach et al., Nature, 290:140 (1981)), lithium acetate method (Okazaki et al., Nucleic Acids Res., 18:6485-6489 (1990)), etc.
If the host is a mammalian cell, such as Chinese Hamster ovary-derived (CHO) cells and human HeLa cells, vectors such as pMSG (Clontech) may be used. Recombinant DNA may be introduced into mammalian cells by calcium phosphate method (Graham et al., Virology, 52:456-467 (1973)), DEAE-dextran method (Sussman et al., Mol. Cell. Biol., 4:1641-1643 (1984)), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), electroporation (Neumann et al., EMBO J., 1:841-845 (1982)), etc. If the host is an insect cell, a baculovirus vector such as pBacPAK8/9 (Clontech) can be used. Transformation of insect cells is done by the methods described in the literature (Bio/Technology 6:47-55 (1980)).
Recombinant proteins expressed in host cells can be purified by known methods. The proteins can also be synthesized as fusion proteins tagged with histidine residues at the N-terminus, or fused to glutathione-S-transferase (GST), and purified by using their binding ability toward a metal chelating resin, or a GST affinity resin (Smith et al., J. Biol. Chem., 263:7211-7215 (1988)), respectively. For instance, when the vector pESP-1 is used, the protein of interest is synthesized as a fusion protein with GST, which can be purified using GST affinity resin. The fusion protein may be digested with thrombin, or blood coagulating factor Xa to liberate the protein of interest.
Moreover, DNA encoding the proteins of the present invention can be used in gene therapy of diseases that arise from a mutation of the protein. When used in gene therapy, the DNA can be introduced into human cells using retrovirus vectors (Danos et al., Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988); Dranoff et al., Proc. Natl. Acad. Sci. USA, 90:3539-3543 (1993)), adenovirus vectors (Wickham et al., Cell, 73:309-319 (1993)), etc. To administer the vector to patients, transplantation of bone marrow, subcutaneous injection, and intravenous injection can be used (Asano S., Protein Nucleic acid and Enzyme, 40:2491-2495 (1995)).
Furthermore, the present invention provides antibodies capable of binding to the proteins of the invention. Antibodies against the proteins can be prepared by known methods in the art (for instance, refer to Shin-Seikagaku-Jikken-Kouza I: Protein I 389-406, Tokyo-Kagaku-Doujin). For instance, polyclonal antibodies are prepared as follows. An appropriate dose of the above proteins or partial peptides thereof are administered into immune animals such as rabbits, guinea pigs, mice, or chickens. Administration may be performed together with an adjuvant (such as FIA or FCA) that promotes antibody production, and usually performed every couple of weeks. The titer of antibodies can be increased by performing multiple immunizations. After the final immunization, antisera are obtained by withdrawing blood from immune animals. Polyclonal antibodies are purified from antisera by ammonium sulfate precipitation, fractionation by anion exchange chromatography, or affinity chromatography with either Protein A or immobilized antigen. Monoclonal antibodies are prepared as follows. The proteins of the invention or partial peptides thereof are administered into immune animals as described above. After the final immunization, their spleens or lymph nodes are excised. Then, antigen-producing cells are recovered from the spleens or the lymph nodes, and fused with myeloma cells using polyethylene glycol and such to produce hybridomas. Desired hybridomas are selected by screening, and their culture supernatant is used to prepare monoclonal antibodies. Monoclonal antibodies can be purified by ammonium sulfate precipitation, fractionation by anion exchange chromatography, or affinity chromatography with either Protein A or immobilized antigen. Antibodies prepared thereby can be used not only in affinity purification of the protein of the invention, but also for the diagnosis or antibody treatment of diseases arising from the abnormal expression of the receptors, or detection of the expression level of the receptors.
If used for antibody treatment, humanized antibodies or human antibodies are preferable. Humanized antibodies, in case of mouse-human chimeric antibodies, are prepared by isolating the gene encoding the antibody against the G protein-coupled receptor protein from the producing mouse cells, replacing the constant region of the H chain of the antibody with that of the human IgE, and introducing it into mouse myeloma J558L cells (Neuberger et al., Nature, 314:268-270 (1985)). Human antibodies can be prepared by immunizing mice, whose immune system is replaced with that of human, with the protein.
Furthermore, the present invention provides a method of screening for ligands or their analogues of the protein of the invention. The methods include such processes as exposing a test compound to the G protein-coupled receptor protein or its partial peptide, and selecting compounds that are capable of binding to the proteins or the peptide. Compounds to be tested include compounds or their analogues such as acetylcholine, adenosine, adrenaline, noradrenaline, angiotensin, bombesin, bradykinin, C5a anaphylatoxin, calcitonin, cannabinoids, chemokines, cholecystokinin, dopamine, endothelin, formylmethionylpeptide, GABA, galanin, glucagon, glutamate, glycopeptide hormone, histamine, 5-hydroxytryptophan, leucotriene, melanocortin, neuropeptide Y, neurotensin, odorant, opioid peptide, opsin, parathyroid hormone, platelet activating factor, prostanoid, somatostatin, tachykinin, thrombin, thyrotropin releasing hormone, vasopressin, and oxytocin (Watson S. and Arkinstall S., G protein Linked Receptor FactsBook, Academic Press (1994)), and also other purified proteins, expressed products of genes (including libraries), extracts of tissues or cells in which the ligand is stipulated to be expressed (the brain, thalamus, and hypothalamus etc.), and the culture medium of the cells. The proteins may be used in a form expressed in desired cells (including transformants genetically engineered to express the proteins) or on the cell surface, in the form of the membrane fractions of the cells, or in a form bound to an affinity column. If necessary, test compounds may be labeled appropriately. Methods for labeling include radioisotope labeling, and fluorescence labeling, but are not limited thereto. The binding between the proteins and test compounds can be examined by detecting the label added to the compound (for instance, measuring the radioactivity or fluorescence intensity), or using as an index, intracellular signaling triggered by the compound binding to the protein (such as G protein activation, the change in the concentration of Ca2+ or cAMP, phospholipase C activation, and the change in pH). Specific methods can be employed as described in the literatures (Cell Calcium, 14:663-671 (1993); Analytical Biochemistry, 226:349-354 (1995); J. Biol. Chem., 268:5957-5964 (1993); Cell, 92:573-585 (1998); Nature, 393:272-273 (1998)), and unexamined published Japanese patent application (JP-A) No. Hei 9-268. Alternatively, the binding may be detected by measuring the activity of a reporter gene using two-hybrid system (Zervos et al., Cell, 72:223-232 (1994); Fritz et al., Nature, 376:530-533 (1995)).
The present invention also provides a method of screening for a compound which can inhibit the binding between the proteins of the invention and their ligands or their analogues. The method includes the steps of (a) exposing the ligand or its analogue to the proteins of the present invention or their partial peptides in the presence of a test compound, and detecting the binding activity between the proteins or partial peptides and the ligand or its analogue, and (b) comparing the binding activity detected in (a) with that in the absence of the test compound, and selecting a compound that reduces the binding activity. Compounds to be tested include proteins, peptides, non-peptide compounds, artificially synthesized compounds, extracts of tissues and cells, sera, but are not limited thereto. The proteins may be used in a form expressed in desired cells (including transformants genetically engineered to express the proteins) or on the cell surface, in a form of the membrane fractions of the cells, or in a form bound to an affinity column. If necessary, ligands may be labeled appropriately. Methods for labeling include radioisotope labeling, and fluorescence labeling, but are not limited thereto. As a ligand, for example, histamine can be preferably used. An analogue of histamine, for example, R(xe2x88x92)-xcex1-methylhistamine, can be used.
The binding activity between the proteins of the present invention or their partial peptides and ligands or their analogues can be examined by detecting a label added to the ligand or its analogue (for instance, measuring the radioactivity or fluorescence intensity), or using cellular change, as an index, that are triggered by the compound binding to the protein (such as G protein activation, the change in the concentration of Ca2+ or cAMP, phospholipase C activation, and the change in pH). Specific methods can be employed by the method of Zlokarmik et al. (Science, 1998, 279:84) as described in examples. Moreover, the methods can be employed as described in the literatures (Cell Calcium, 14:663-671 (1993); Analytical Biochemistry, 226:349-354 (1995); J. Biol. Chem., 268:5957-5964 (1993); Cell, 92:573-585 (1998); Nature, 393:272-273 (1998)), and JP-A No. Hei 9-268). If the results of the detection show that the binding activity in the presence of a test compound is lower than that in the absence of the compound (control), the compound is judged to be capable of inhibiting the binding between the proteins or their partial peptides and the ligands or their analogues. These compounds include those capable of triggering the intracellular signaling through binding to the protein (agonist), and those not having such activity (antagonist). Agonists have similar bioactivities to those of the ligands of the proteins. On the other hand, antagonists inhibit the bioactivities of the ligands. Therefore, these agonists and antagonists are useful as medicinal compositions for treatment of diseases arising from disorders in the signaling pathway mediated by the proteins.
In addition, the present invention provides a method of screening for a compound which inhibits or promotes an activity of the protein of the present invention. The screening method contains the steps of (a) exposing a ligand for the protein or an analogue thereof to cells expressing the protein in the presence of a test compound, (b) detecting a change in cells associated with the binding of the protein to the ligand or the analogue thereof, and (c) selecting a compound which inhibits or promotes the change in the cells detected in (b) in comparison with the change in the cells in the absence of the test compound. Compounds to be tested include proteins, peptides, non-peptide compounds, artificially synthesized compounds, extracts of tissues and cells, and sera, but are not limited thereto. A compound isolated by the screening in which the inhibition of the above binding activity is an index can be used as a test compound. Cells which express the proteins of the present invention can be prepared by, for example, inserting a DNA encoding the proteins to an appropriate vector, and introducing the vector into an appropriate animal cell, as described in Example 5. Into the expression vector, a marker gene for selecting a recombinant may be inserted. As a ligand for stimulating the proteins of the present invention, for example, histamine can be preferably used. An analogue of histamine, for example, R(xe2x88x92)-xcex1-methylhistamine, can be used.
A change in cells associated with the binding of a ligand or an analogue thereof to the proteins of the present invention can be detected, for example, using, as an index, an activation of G protein, a concentration change of Ca2+ or cAMP, an activation of phospholipase C, a change of pH. Specific methods can be employed by the method of Zlokarmik et al. (Science, 1998, 279:84) as described in Example 6. Moreover, the methods can be employed as described in the literatures (Cell Calcium, 14:663-671 (1993); Analytical Biochemistry, 226:349-354 (1995); J. Biol. Chem., 268:5957-5964 (1993); Cell, 92:573-585 (1998); Nature, 393:272-273 (1998)), and JP-A No. Hei 9-268).
As a result of this detection, when a test compound used inhibits a change in cells in comparison with that in cells in case of reacting a ligand or an analogue thereof in the absence of the test compound, the used test compound is judged to be a compound which inhibits an activity of the proteins of the present invention. In contrast, when a test compound enhances a change in the cells, the compound is judged to be a compound which promotes an activity of the protein of the present invention.
A compound isolated by the screening method of the present invention (an agonist or antagonist of the proteins of the present invention) can be applied to, for example, attention deficit hyperactivity disorder, Alzheimer""s disease, memory disorder, dysgnosia, schizophrenia, sleep disorder, insomnia, sleep-induced apnea syndrome, narcolepsy, articular rheumatism, osteoarthritis, gastric ulcer, inflammatory intestine disorder, ischemic heart disease, arrhythmia, high or low blood pressure disorder, epilepsy, obesity, cibophobia, depression, anxiety, migraine, asthma, Huntington""s disease, pain, nicotine abstinence symptoms (Trends in Pharmacological Science, 19:177-183; Stark et al., Drugs of the Future 21:507-520 (1996); Onodera et al., Jpn J. Psychopharmacol., 15:87-102 (1995)). When using these compounds as a drug, the isolated compound itself can be directly administered to the patient, or it can be given after formulating as pharmaceutical compositions by using commonly known pharmaceutical preparation methods. The compound may be administered after formulating by mixing with, for example, pharmaceutically acceptable carriers or media, and specifically, sterilized water, physiological saline, plant oils, emulsifiers, suspending agents, surfactants, stabilizers, binders, lubricants, sweeteners, flavors, coloring agents, and so on. The administration to patients is done by methods commonly known to those skilled in the art, such as intraarterial, intravenous, or subcutaneous injections and, in addition, intranasal, bronchial, intramuscular, or oral administrations. One skilled in the art can suitably select the dosage according to the body-weight or age of a patient, or the method of administration.
Furthermore, the present invention provides a kit for the screening described above, comprising the proteins of the present invention or their partial peptides. The proteins or their partial peptides may be in a form expressed in desired cells (including transformants genetically engineered to express the protein) or on the cell surface, in a form of membrane fractions of the cell, or in a form bound to an affinity column. Components of the kit of the invention may include, other than the above-described receptor protein samples, ligand samples (both labeled and unlabeled), and buffers for the reaction between the ligand and the receptor protein, and wash solutions. Labels to be added to the ligands include radioisotope and fluorescence, for instance. The kit of the invention can be used as described in JP-A No. Hei 9-268. Moreover, for example, in the screenings using the detection system of the cAMP concentration change described in Example 6, and the detection system of the binding activity described in Example 7, the kit of the present invention can be used.
All references and patents cited herein are incorporated by reference in their entirety.